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目的 研究幽门螺杆菌(Helicobacter pylori,H.pylori)lpp20基因与麦芽糖结合蛋白基因的融合表达产物的纯化方法,为幽门螺杆菌基因工程疫苗的制备建立基础。方法 采用本研究室分离的HpMEL-HP27菌株提取染色体DNA,用PCR方法从HP染色体DNA上扩增出lpp20基因片段,将目的基因插入到表达载体pMAL-c2X中,用重组质粒转化大肠杆菌(E.coli TB1)。采用IPTG进行诱导表达。用多糖树脂(amylose resin)作为填充料,制备层析柱,将可溶性菌体蛋白进行亲和层析。应用SDS-PAGE方法对纯化产物进行分析。结果 融合蛋白的分子量约为60kDa,融合蛋白的表达量约占全菌总蛋白的34％;纯化后的融合蛋白纯度达90％以上。结论 与麦芽糖结合蛋白基因融合的lpp20基因在E.coli TB1中能够高效表达;用多糖树脂作为填充料,进行亲和层析的方法,具有良好的纯化效果。
Objective To study the method for purifying Helicobacter pylori (Hpylori) lpp20 gene and maltose binding protein (EGFP) gene and to establish a foundation for the preparation of genetically engineered Helicobacter pylori vaccine. Methods Chromosomal DNA was extracted from HpMEL-HP27 strain isolated in this research laboratory. The lpp20 gene fragment was amplified from HP chromosome DNA by PCR. The target gene was inserted into expression vector pMAL-c2X. The recombinant plasmid was transformed into E. coli .coli TB1). Induction of expression using IPTG. Using amylose resin as a filler, a chromatographic column was prepared and the soluble bacterial cell proteins were subjected to affinity chromatography. The purified product was analyzed by SDS-PAGE. Results The molecular weight of the fusion protein was about 60 kDa. The fusion protein accounted for about 34% of the total bacterial protein. The purity of the fusion protein was over 90%. Conclusion The lpp20 gene fused with maltose binding protein gene can be highly expressed in E.coli TB1. The method of affinity chromatography using polysaccharide resin as filler has good purification effect.